1. Field of the Invention
The present invention relates to a novel DNA fragment carrying a toluene monooxygenase gene, a novel recombinant DNA containing the DNA fragment, a transformant containing the recombinant DNA, and a method for degrading chlorinated aliphatic hydrocarbon compounds such as trichloroethylene (TCE) and dichloroethylene (DCE) and aromatic compounds such as toluene, benzene, phenol, and cresol. The present invention also relates to a method for environmental remediation useful for cleaning of aqueous media such as wastewater and effluent containing at least either a chlorinated aliphatic hydrocarbon compound or an aromatic compound and air (gas phase) and soil polluted with chlorinated aliphatic hydrocarbon compounds.
2. Related Background Art
Recently, it has become a serious problem the environmental pollution with volatile organic chlorinated compounds which are harmful to the organisms and hardly degradable. Especially, the soil in the industrial areas in Japan as well as abroad is considered to be contaminated with chlorinated aliphatic hydrocarbon compounds such as tetrachloroethylene (PCE), trichloroethylene (TCE), and dichloroethylene (DCE) and aromatic compounds such as toluene, benzene, phenol, and cresol. In fact, there have been a number of reports on actual detection of such pollutants through environmental surveys. It is supposed that these compounds remaining in soil dissolve in ground water via rainwater, and thereby spread over the surrounding areas. There is a strong suspicion that these compounds are carcinogens, and further, these are quite stable in the environment; therefore contamination of groundwater, which is used as a source of drinking water, has become a serious social problem. Therefore, cleaning of soil and aqueous media such as contaminated groundwater by removal and degradation of these compounds and accompanying cleaning of the surrounding gas phase is quite important in view of the environment protection, and technologies for remedying the environment (for example, adsorption treatment using activated carbon, degradation treatment using light and heat) have been developed. Current technologies, however, are not always practical in terms of cost and operability. Recently, microbial degradation of chlorinated aliphatic hydrocarbon compounds such as TCE that is stable in environment has been reported. The microbial degradation method has advantages such as: (1) degradation of chlorinated aliphatic hydrocarbon compounds into harmless substances by using appropriately selected microorganism; (2) no requirement for any special chemicals in principle; and (3) reduction of the labor and costs of maintenance.
The examples of microorganisms capable of degrading TCE are as follows:
Welchia alkenophila sero 5 (U.S. Pat. No. 4,877,736, ATCC 53570, Welchia alkenophila sero 33 (U.S. Pat. No. 4,877,736, ATCC 53571), Methylocystis sp. Strain M (Agric. Biol. Chem., 53, 2903 (1989), Biosci. Biotech. Bichem., 56, 486 (1992), ibid. 56, 736 (1992)), Methylosinus trichosporium OB3b (Am. Chem. Soc. Natl. meet. Div. Environ. Microbiol., 29, 365 (1989), Appl. Environ. Microbiol., 55, 3155 (1989), Appl. Biochem. Biotechnol. 28, 877 (1991), Japanese Patent Application Laid-Open No. 2-92274 specification, Japanese Patent Laid-Open Application No. 3-292970), Methylomonas sp. MM2 (Appl. Environ. Microbiol., 57, 236 (1991), Alcaligenes denitrificans ssp. Xylosoxidans JE75 (Arch. Microbiol., 154, 410 (1990), Alcaligenes eutrophus JMP134 (Appl. Environ. Microbiol., 56, 1179 (1990), Alcaligenes eutrophus FERM-13761 (Japanese Patent Laid-Open Application No. 7-123976), Pseudomonas aeruginosa J1104 (Japanese Patent Application Laid-Open No. 7-236895), Mycobacterium vaccae J0B5 (J. Gen. Microbiol., 82, 163 (1974), Appl. Environ. Microbiol., 55, 2960 (1989), ATCC 29678), Pseudomonas putida BH (Gesuidou Kyoukai-shi (Japan Sewage Works Association Journal), 24, 27 (1987)), Pseudomonas sp. strain G4 (Appl. Environ. Microbiol., 52, 383 (1968), ibid. 53, 949 (1987), ibid. 54, 951 (1988), ibid. 56, 279 (1990), ibid. 57, 193 (1991), U.S. Pat. No. 4,925,802, ATCC 53617, this strain was first classified as Pseudomonas cepacia and then changed to Pseudomonas sp.), Pseudomonas mendocia KR-1 (Bio/Technol.; 7, 282 (1989)), Pseudomonas putida F1 (Appl. Environ Microbiol., 54, 1703 (1988), ibid. 54, 2578 (1988)), fluorescens PFL12 (Appl. Environ. Microbiol., 54, 2578 (1988)), Pseudomonas putida KWI-9 (Japanese Patent Application Laid-Open No. 6-70753), Burkholderia cepacia KK01 (Japanese Patent Application Laid-Open No. 6-22769), Nitrosomonas europaea (Appl. Environ. Microbio., 56, 1169 (1990), Lactobacillus vaginalis sp. nov (Int. J. Syst. Bacteriol., 39, 368 (1989), ATCC 49540), Nocardia corallina B-276 (Japanese Patent Application Laid-Open No. 8-70881, FERM BP-5124, ATCC 31338), and so on.
The problem in actually using these degrading microorganisms in environmental remediation treatment, however, resides in optimizing and maintaining expression of their degradation activity for chlorinated aliphatic hydrocarbon compounds such as TCE. In an environmental remediation treatment which utilizes phenol, toluene, methane, or the like as an inducer, continuous supply of the inducer is indispensable, since depletion of such inducers directly results in stoppage of degradation of chlorinated aliphatic hydrocarbon compounds. Presence of such inducers, on the other hand, may inhibit the efficient degradation of the target substance such as TCE, since the affinity of the chlorinated aliphatic hydrocarbon compounds such as TCE as a substrate is considerably low in comparison with these inducers. In addition, precise control of the inducer concentration on the treatment spot is difficult.
Thus, use of an inducer is a large problem in practical application of environmental remediation treatment utilizing microorganisms.
In order to solve the problem, Nelson et al. developed a method using tryptophan as an inducer for degradation of volatile organic chlorinated compounds (Japanese Patent Application Laid-Open No. 4-502277). Tryptophan, however, is a very expensive substance, and although tryptophane has no toxicity or risk as a substance, it is not preferable to introduce excessive carbon and nitrogen sources into environment since it may induce eutrophication. In addition, the problem that tryptophan serves as a competitive inhibitor in degradation of TCE still remains.
Shields et al. obtained a mutant strain of Pseudomonas cepacia G4 (changed to Pseudomonas sp. upon deposition to ATCC) by the transposon technique, which mutant strain does not require an inducer (in this case, phenol or toluene) and can degrade TCE (Appl. Environ. Microbiol., 58, 3977 (1992), International Publication No. WO/19738). Also, a mutant not requiring methane as the inducer has been isolated from Methylosinus trichosporium OB3b, a methanotroph capable of degrading TCE (U.S. Pat. No. 5,316,940).
Japanese Patent Application Laid-Open No. 8-294387 also discloses strain JM1 (FERM BP-5352) capable of degrading volatile organic chlorinated compounds and aromatic compounds without requiring an inducer, isolated by nitrosoguanidine mutagenization of strain J1 (FERM BP-5102). While, it has been studied to introduce resting cells expressing TCE-degrading activity into the remediation site after the preculture of the cells in the presence of an inducer (Environ. Sci. Technol., 30, 1982 (1996)).
It has been reported that remediation treatment not requiring the inducer actually makes the remediation treatment easy and efficient compared to the conventional treatment using inducers.
However, the growth control of the degrading microorganisms is very important for both the expression of the degradation activity on demand and the continuation of degradation. When resting cells are used, it is a problem to be solved that TCE cannot be degraded beyond the amount and period of degradation capacity of the introduced resting cells. In addition, in a large scale treatment, there are further problems that degradation activity will decrease since it takes a long time to prepare resting cells; the treating apparatus must be large in scale; treatment process is complicated; and the cost may be unfavorably high. Accordingly, it has been attempted to introduce a plasmid carrying a DNA fragment containing a gene region encoding oxygenase or hydroxylase into a host microorganism to make the host express the TCE degradation activity constitutively or inducibly using a harmless inducer. For example, there are Pseudomonas mendocina KR-1 (Japanese Patent Application Laid-Open No. 2-503866, Pseudomonas putida KWI-9 (Japanese Patent Application Laid-Open No. 6-105691), Pseudomonas putida BH (Summary of 3rd Conference on Pollution of Ground Water/Soil and Its Protective Countermeasure, p.213 (1994)), and a transformant carrying both a toluene degradation enzyme gene derived from Pseudomonas putida F1 and a biphenyl degradation enzyme gene derived from Pseudomonas pseudoalkaligenes (Japanese Patent Application Laid-Open No. 7-143882). However, the reported TCE degradation activity of the transformants are low, and the advantages of the transformants has not been fully exploited for efficient degradation of TCE, such as the ease of degradation control, freedom in designing recombinant, and no requirements for inducers, far from efficient TCE degradation.